METHODS OF SCREENING OF PP1-INTERACTING POLYPEPTIDES OR PROTEINS, PEPTIDES INHIBITING PP1c BINDING TO Bcl-2 PROTEINS, BCL-XL AND BCL-W, AND USES THEREOF

ABSTRACT

The invention relates to methods for identifying novel PP1-interacting polypeptides and proteins, compounds which are able to inhibit the binding of PP1c to certain factors naturally interacting with it, especially proteins of the Bcl-2 family (such as Bcl-x L  and Bcl-w).

BACKGROUND OF THE INVENTION

The invention relates to methods for identifying novel PP1-interactingpolypeptides and proteins, compounds which are able to inhibit thebinding of PP1c to certain factors naturally interacting with it,especially proteins of the Bcl-2 family (such as Bcl-x_(L) and Bcl-w),and pharmaceutical compositions comprising the same.

PRIOR ART

The serine/threonine phosphatases are classified as type 1 (PP1) or type2 (PP2), depending on their substrate specificity and sensitivity toinhibitors. PP1 regulates cell cycle progression, proliferation,transcription, protein synthesis, cytokinesis and neuronal signaling (McAvoy et al, 2001). PP1 is regulated by its interaction with a variety ofprotein subunits that target the catalytic subunit (PP1c) to specificsubcellular compartments and determines its localization, activity andsubstrate selectivity (Bollen et al, 2001). PP1 can be regulated by theinteraction between a catalytic subunit and multiple targeting subunitsthat allow specific dephosphorylation of diverse cellular targets. Thenumber of known PP1c targeting subunits is continuously increasing andto date nearly thirty unique mammalian proteins have been alreadyidentified. PP1 interacting protein include the glycogen-bindingsubunits, RGL/GM, GL, PTG/R5/U5, and R6 which target the phosphatase toglycogen, the myosin-associating subunits, M110, NIPP-1, p99/PNUTS, andSds22, which may direct the phosphatase to the nucleus (Bollen, 2001).Previous studies based on structural and X-ray crystallography analysisof PP1 interacting proteins indicated that PP1c binds to distinct knowninteracting proteins through a short amino-acid sequence. The [RK]VxF(or [RK]xVxF) motifs represent a widespread consensus sequence for therecognition and binding of distinct regulatory subunits and interactingproteins with PP1c (Egloff et al, 1997; Aggen et al, 2000).

Using a murine T cell line that can be propagated independently in thepresence of IL-2 or IL-4 (Pitton et al, 1993), the inventors havedescribed ‘ that PP1c is a Ras-activated phosphatase thatdephosphorylates Bad (a pro-apoptotic member of the Bcl-2 proteinfamily) prior to induce apoptosis in response to IL-2 deprivation(Ayllón et al, 2000). By performing biochemichal competitive studies,the inventors also recently identified Bcl-2 as a new targeting subunitof PP1c that controls its association to Bad in IL-2-stimulated cells(Ayllón et al, 2001).

The Bcl-2 family proteins act as an intracellular checkpoint in theapoptotic pathway. The Bcl-2 family of proteins is divided into twofunctional groups: anti-apoptotic members such as Bcl-2, Bcl-x_(L),Bcl-w, A1 and Mcl-1 and pro-apoptotic members such as Bax, Bak,Bcl-x_(s) as well as the BH3-only member Bad (White, 1996; Reed, 1998;Chao and Korsmeyer, 1998). Balance between homo- and hetero-dimers ofBcl-2 family members may be critical to maintain cell proliferation orapoptosis (Jacobson, 1997; Korsmeyer, 1999; Gross et al, 1999). Up- ordown-regulation of these proteins may account for survival of some celltypes, although it is also possible that survival factors use proteinkinases or phosphatases to alter the ability of these proteins topromote cell survival or apoptosis. Anti-apoptotic Bcl-2 family membersinteract with other death agonists of the Bcl-2 family and withnon-Bcl-2 family proteins, including R-Ras, H-Ras, Raf, caspases,calcineurin and the serine/threonine phosphatase PP1c (Rebollo et al,1999; Ayllón et al, 2001). The Bcl-2 family has been defined by sequencehomology based upon specific conserved motifs termed Bcl-homologyregions (BH1, BH2, BH3 and BH4 domains). BH1, BH2 and BH3 domains havebeen shown to be important in homodimerization or heterodimerization andin modulating apoptosis. Anti-apoptotic molecules have a specific BH4domain.

Bad shares identity only in the BH3 domain (Zha et al, 1997) and formshetero-dimers with Bcl-2 and Bcl-x (Ottilie et al, 1997). Uponstimulation of cells with IL-3, NGF and GM-CSF, Bad becomes serinephosphorylated (Del Peso et al, 1997; Datta et al, 1997), resulting inassociation to the 14-3-3 protein and abolishing interaction with Bcl-x(Hsu et al, 1997). It has been recently shown that association of 14-3-3protein to Bad is dependent on serine 155 phosphorylation of Bad (Dattaet al, 2000; Zhou et al, 2000).

Bcl-w is a pro-survival protein bearing the four conserved Bcl-2homology (BH) domains (Gibson et al, 1996). Enforced expression ofBcl-w, like Bcl-2, renders lymphoid and myeloid cell lines resistant toapoptosis induced by cytokine deprivation. The anti-apoptotic moleculeBcl-x_(L) also contains the four BH conserved domains (Núnez et at,1994). A second Bcl-x isoform, Bcl-x_(s), encodes a smaller protein of170 amino acids that enhances apoptosis (Minn et al, 1996). Bcl-x_(L)contains a hydrophobic segment at the C-terminal end that is believed toserve as a membrane anchor (Boise et al, 1993).

Apoptosis or programmed cell death is an active process in which cellsinduce their self-destruction in response to specific cell death signalsor in the absence of cell survival signals. This active process, isactually essential in the normal development and homeostasis ofmulticellular organisms. It is opposed to necrosis which is cell deathoccurring as a result of severe injurious changes in the environment.

Various pathologies occur due to a defective or aberrant regulation ofapoptosis in the affected cells of an organism. For example, defectsthat result in a decreased level of apoptosis in a tissue as compared tothe normal level required to maintain the steady-state of the tissue canpromote an abnormal increase of the amount of cells in a tissue. Thishas been observed in various cancers, where the formation of tumorsoccurs because the cells are not dying at their normal rate. Some DNAviruses such as Epstein-Barr virus, African swine fever virus andadenovirus, also inhibit or modulate apoptosis, thereby repressing celldeath and allowing the host cell to continue reproducing the virus.

To the contrary, a defect resulting in an increase of cell death in atissue may be associated with degenerative disorders wherein cells aredying at a higher rate than they regenerate. This is observed in variousdisorders, such as AIDS, senescence, and neurodegenerative diseases.

Compounds that modulate positively or negatively apoptosis can providemeans for the treatment or the prevention of these disorders. As aconsequence, the delineation of apoptotic pathways provides targets forthe development of therapeutic agents that can be used to modulate theresponse of a cell to apoptotic or cell survival signals.

The results disclosed in the present invention indicate that theanti-apoptotic: members of the Bcl-2 family, Bcl-w and Bcl-x_(L) arealso targeting subunits of PP1c in IL-4-stimulated cells. Thisobservation offers a way to a novel general mechanism of regulation ofcell apoptosis that may play a role in the regulation of pro- oranti-apoptotic molecules in response to cell death or cell survivalsignals. The invention is therefore of a particular importance in thefields of cancer therapy and neurodegenerative diseases therapy.

More generally, the present invention provides means to modulate theinteraction between PP1 and the proteins or polypeptides that bind toit. Therefore, the present invention has applications in a wide range offields, since PP1 is involved in many biological pathways. For example,it is known that a decrease in phosphorylation of the PP1 complexactivates the smooth muscle myosin light chains and hence relaxes smoothmuscles (see, Uehata et al., 1997). High blood pressure could hence be atarget of the present invention.

PP1 is a major eukaryotic protein serine/threonine phosphatase thatregulates diverse cellular processes such as cell cycle, transcriptionand protein synthesis.

PP1-regulation can also effect the downstream regulation of hepaticglycogen synthesis which in turn would lower blood glucose levels andthus treat diabetes in which hyperglycemia is a severe problem.

Moreover, PP1 is involved in several bacterial, viral and parasiticinfections, and inhibiting its interaction with some of its partners inan infectious context could be beneficial to the patient.

SUMMARY OF THE INVENTION

To be concise, the following Summary, the Preferred Embodiments and theExamples of the present invention are described below, it beingunderstood that literal word-for-word description of all of theembodiments and combinations thereof would be recognized by the personskilled in the art and hence, literally repetiveness not beingnecessary. It should be appreciated that the Examples, as well as thepreferred embodiments, the Summary of the Invention and certain aspectsof the prior art, can be combined in all variations; one aspect of theinvention combined with another irregardless of their place in thisdescription of the entire specification, without deviating from thepresent invention, as recognized by the person skilled in the art,without any limitations.

Thus, the present invention relates to a peptide or a set of peptideswhich mimicks both the motifs M1 and M2 and which is able to inhibit theBcl-x_(L)/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c interactions,wherein the motif M1 has the sequence FXX[RK]X[RK], and the motif M2 hasthe sequence [RK]VX[FW] or [RK]XVX[FW], wherein X is any amino acid.

In another embodiment, the present invention relates to a set ofpeptides comprising R (NWGRIVAFFSF) and F (GDEFELRYRRAF) peptides.

In yet another embodiment the present invention relates to apharmaceutical composition comprising a peptide or a set of peptideswhich mimicks both the motifs M1 and M2 and which is able to inhibit theBcl-x_(L)/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c interactions. Sucha composition can, for example, comprise R (NWGRIVAFFSF) and F(GDEFELRYRRAF) peptides. Peptides with modified amino acids(glycosylation, acetylation, phosphorylation, amidation or derivation byknown protecting/blocking groups) can also be used in the compositionsaccording to the invention.

In yet another embodiment the present invention relates to apharmaceutical composition comprising a vector comprising a nucleic acidencoding a peptide or a set of peptides which mimicks both the motifs M1and M2.

In another embodiment the present invention relates to a method ofidentifying a PP1-interacting polypeptide or protein, comprisingdetecting in the sequence of a polypeptide or protein, the presence oftwo PP1-binding motifs M1 and M2.

In yet another embodiment the present invention discloses a method ofscreening compounds that interact with PP1 regulators, wherein peptideswhich mimick both the motifs M1 and M2 and which are able to inhibit theBcl-x_(L)/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c interactions, areimmobilized on a support and the interaction of said compounds with saidpeptides are tested.

In yet another embodiment, the present invention relates to a method ofscreening compounds that interact with PP1. This method comprisesobtaining antibodies to peptides which mimick both the motifs M1 and M2and which are able to inhibit the Bcl-x_(L)/PP1c, the Bcl-w/PP1c, or theBcl-2/Bad/PP1c interactions, and testing the interaction of saidcompound with said antibodies.

In another embodiment, the present invention relates to a method forinhibiting in vitro the interaction between PP1c and Bcl-x_(L) or Bcl-w,comprising a step of adding a peptide or a set of peptides which mimicksboth M1 and M2.

In the above methods, the peptides which mimick both the motifs M1 andM2 and which are able to inhibit the Bcl-x_(L)/PP1c, the Bcl-w/PP1c, orthe Bcl-2/Bad/PP1c interaction can be for example the R (NWGRIVAFFSF)and F (GDEFELRYRRAF) peptides.

In yet another embodiment, the present invention relates to a kitcomprising peptides which mimick both the motifs M1 and M2 and which areable to inhibit the Bcl-x_(L)/PP1c, the Bcl-w/PP1c, or theBcl-2/Bad/PP1c interactions. Such a kit can, for example, comprise R(NWGRIVAFFSF) and F (GDEFELRYRRAF) peptides. These peptides may beimmobilzed on a solid support.

In yet another embodiment the present invention relates to a kitcontaining antibodies to peptides which mimick both the motifs M1 and M2and which are able to inhibit the Bcl-x_(L)/PP1c, the Bcl-w/PP1c, or theBcl-2/Bad/PP1c interactions. Said antibodies can for example be raisedagainst R (NWGRIVAFFSF) and F (GDEFELRYRRAF) peptides.

In yet another aspect, the present invention relates to a method fortreating animals and vegetables that have any disease involved in thePP1c pathway, comprising administering to animals and vegetables in needof such treatment peptides which mimick both the motifs M1 and M2 andwhich are able to inhibit the Bcl-x_(L)/PP1c, the Bcl-w/PP1c, or theBcl-2/Bad/PP1c interactions.

In yet another aspect, the present invention relates to a method fortreating an animal that has diabetes or hypertension or a neurologicaldisorder or a viral infection or a parasitic infection, comprisingadministering to an animal in need of such treatment peptides whichmimick both the motifs M1 and M2 and which are able to inhibit theBcl-x_(L)/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c interactions.

In the above methods, R (NWGRIVAFFSF) and F (GDEFELRYRRAF) peptides canfor example be administered.

In another embodiment, the present invention relates to the use ofpeptides which mimick both the motifs M1 and M2 and which are able toinhibit the Bcl-x_(L)/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1cinteractions, to treat diabetes or hypertension or neurologicaldisorders or a viral infection or a parasitic infection.

In yet another embodiment the present invention relates to the use ofpeptides which mimick both the motifs M1 and M2 and which are able toinhibit the Bcl-x_(L)/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1cinteractions, for the preparation of a medicament to treat diabetes orhypertension or neurological disorders or a viral infection or aparasitic disease. R (NWGRIVAFFSF) and F (GDEFELRYRRAF) peptides can forexample be used according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Association of Bcl-x_(L) and Bcl-w to PP1c and Bad.

A) Cytoplasmic extracts from IL-4-stimulated (60 U/ml) or -deprivedcells were immunoprecipitated with anti-Bcl-x_(L) or anti-Bcl-wantibody, transferred to nitrocellulose and blotted with anti-Bad,anti-PP1c, anti-Bcl-x_(L) and anti-Bcl-w antibody. Protein bands weredetected using ECL system. Molecular weights of the correspondingproteins is shown. Similar results were obtained in three independentexperiments. B) Cytoplasmic extracts from IL-4-stimulated cells wereimmunoprecipitated with anti-p55 IL-2R chain antibody, transferred tonitrocellulose and blotted with anti-Bcl-w, Bcl-x_(L) or anti-p55 IL-2Rantibodies. Protein bands were detected using the ECL system. C)Cytoplasmic extracts from freshly isolated thymocytes wereimmunoprecipitated with anti-Bad, anti-PP1c or an irrelevant serum andblotted with anti-Bcl-2, anti-Bcl-x_(L), anti-Bad and anti-PP1c.Proteins were detected as in A. Similar results were obtained in threeindependent experiments.

FIG. 2. Effect of IL-4-deprivation on Bad, PP1c, Bcl-x_(L) and Bcl-wexpression.

A) Ts1αβ cells were IL-4-stimulated or -deprived for the timesindicated, then lysed. Proteins were transferred to nitrocellulose andprobed with anti-Bcl-x_(L), anti-Bcl-w, anti-Bad and anti-PP1cantibodies. Similar results were obtained in two independentexperiments.

B) Cytoplasmic extracts from IL-4-stimulated or 24 h-deprived cells wereimmunoprecipitated with anti-Bcl-x_(L) or anti-Bcl-w, separated in agradient SDS-PAGE gel and blotted with anti-Pser, anti-Bad, anti-PP1cand, as internal control, with anti-Bcl-x_(L) and Anti-Bcl-w. Similarresults were obtained in three independent experiments.

C) Cytoplasmic extracts from control, IL-4 stimulated or deprived cells(1×10⁷) were immunoprecipitated with anti-Bad antibody and blotted withanti-Bad serine 112, serine 136 and serine 155. As internal control, theblot was developed with anti-Bad antibody. Similar results were obtainedin two independent experiments. Positive control for serine 112 and 136phosphorylation of Bad, IL-2 stimulated cells (lane C); positive controlfor serine 155 phosphorylation of Bad, Bad-transfected COS cells (laneC). D) Total extracts (T) of cytoplasmic lysates from IL-4-stimulated ordeprived cells (1×10⁷) were immunoprecipitated with anti-PP1c, anti-Raf,anti-Bcl-x_(L) or anti-Bad antibody and blotted with anti-14-3-3,anti-PP1c, anti-Bcl-x_(L) and anti-Bad. Similar results were obtained intwo independent experiments.

FIG. 3. Estimation of serine/threonine phosphatase activity in controlor OA-treated Bcl-x, Bad and Bcl-w immunoprecipitates.

A) Phosphatase activity was estimated in Bad, Bcl-x_(L) and Bcl-wimmunoprecipitates from IL-4-stimulated cells using ³²P phosphorylase aas substrate.

B) Different concentrations of OA were added to Bad or Bcl-wimmunoprecipitates from IL-4 stimulated cells. Phosphatase activity wasestimated using ³²P phosphorylase a as substrate. The reaction was as inA. Similar results were obtained in three independent experiments.Phosphatase activity is represented as the percentage of maximalactivity in untreated supernatants.

FIG. 4. Estimation of serine/threonine phosphatase activity afterBcl-_(L) and Bcl-w depletion.

A) Bcl-x_(L) and Bcl-w were depleted from cytoplasmic lysates of IL-4stimulated cells by four sequential immunoprecipitations. Phosphataseactivity was estimated in Bad immunoprecipitates from controlIL-4-stimulated cells or in Bad immunoprecipitates depleted of Bcl-x_(L)and Bcl-w after four sequential immunoprecipitations. Phosphataseactivity is represented as the percentage of the maximal activitydetected in control anti-Bad immunoprecipitates. B) The effect ofBcl-x_(L) and Bcl-w depletion in PP1c/Bad association was analyzed.Cytoplasmic extracts from control IL-4-stimulated cells or Bcl-x_(L),and Bcl-w depleted cytoplasmic extracts were immunoprecipitated withanti-Bad or anti-PP1c antibody and blotted with anti-Bad, anti-Bcl-w,anti-Bcl-x_(L) and anti-PP1c. Similar results were obtained in threeindependent experiments. Protein bands were detected using the ECLsystem.

FIG. 5. PP1c binding assay on cellulose-bound Bcl-x_(L) or Bcl-wpeptides. A) Sequence of F X X R X R motif of Bcl-2, Bcl-x_(L) andBcl-w. B) Membrane with Bcl-x₁ or Bcl-w peptides containing the R/K XV/I X F or F X X R X R motif, as well as peptides containing mutatedmotifs were incubated with purified PP1c, followed by anti-PP1c antibodyand PO-conjugated secondary antibody. Spots were detected using ECLsystem. The R/K X V/I X F and F X X R X R motifs are in bold. Mutatedamino acids into the motif are in bold and underlined. Similar resultswere obtained in two independent experiments. Peptide 1 corresponds tothe PP1 binding motif of Bcl-x_(L) and Bcl-w. Peptide 2 corresponds tothe mutated PP1 binding site were V and F residues were mutated to A.

FIG. 6. Effect of R, R*, F and R+F peptides on the interactionBcl-x_(L)/PP1c/Bad and Bcl-w/PP1c/Bad.

A) Cytoplasmic extracts from control IL-4-stimulated cells wereimmunoprecipitated with anti-Bad antibody. The interactionBcl-x_(L)/PP1c/Bad and Bcl-w/PP1c/Bad was competed with 1.5 mM of R, R*,F or R+F peptides for 30 min at room temperature. Immunoprecipitateswere washed, transferred to nitrocellulose and blotted with anti-Bad,anti-PP1c, anti-Bcl-x_(L) and anti-Bcl-w. Similar results were obtainedin two independent experiments. For sequence of peptides, see Materialsand Methods or FIG. 5A and 5B.

B) Cytoplasmic lysates from IL-4-stimulated cells wereimmunoprecipitated with anti-Bad antibody. Immunoprecipitates weretreated with 1.5 mM of R, R*, F or R+F peptides for 30 min at roomtemperature. Immunoprecipitates were washed and phosphatase activityestimated using ³²P phosphorylase a as substrate. Similar results wereobtained in two independent experiments.

C) Cytoplasmic lysates from IL-4-stimulated cells wereimmunoprecipitated with anti-Bad antibody and then treated with 1.5 mMof R+F peptide or 3 mM of R or F peptide (30 min, room temperature).Immunoprecipitates were washed and phosphatase activity estimated as inB.

FIG. 7. Effect of OA and antisense oligonucleotides on apoptosis andserine 136 phosphorylation of Bad.

A) Cells were treated with or without 1 μM OA in the presence or theabsence of IL-4. Cytoplasmic lysates were immunoprecipitated withanti-Bad antibody, transferred to nitrocellulose and probed withphospho-Bad ser 136 and anti-Bad, the latter to verify that OA treatmentin vivo does not affect Bad expression. Protein bands were detectedusing ECL.

B) Cells were treated for 6 h with or without 1 μM OA in the presence orabsence of IL-4 and then washed, stained with ptopidium iodide andanalyzed by flow cytometry.

C) Cells were treated for 24 h with or without 15 μM sense or antisenseoligonucleotide in the presence or the absence of IL-4. Oligonucleotideswere added a 0, 12 and 18 h and then cells were washed, stained withpropidium iodide and analyzed by flow cytometry. The expression ofBcl-x_(L) and Bcl-w upon sense and antisense treatment was analyzed bywestern blot.

FIG. 8: A) Two putative PP1 binding motifs in Bcl-2 proteins Sequencealignment in the vicinity of BH1 and BH3 domain of some Bcl-2 proteins.These sequences are perfectly conserved in various species (human,mouse, rat, bovine . . . ).

B) A role for Serine phosphorylation in PP1 binding

Six peptides (of 14 AA length) were synthesized and covalenty linked toa cellulose membrane prior to be analyzed for PP1-binding as described.A punctual AA mutation in Ser-136 was introduce in 4 peptides (mutant2,3,5,6).

C) Association of P13-K p85 (lane 1), P13-K p110 (lane 2), HSP70, (lane3),and CD4 (lane 4) to PP1c.

IL-4 treated TS1 αβ cells (1×10⁷) were used for immunoprecipitation asusually described. Immunoprecipitates were transferred tonitrocellulose, blocked and incubated with anti-PP1c primary antibody.Membrane was washed and incubated with PO-conjugated secondary antibodyand proteins were developed using the ECL.

FIG. 9: Histogram to illustrate the number of proteins and the number ofamino-acids distancing the F-x-x-[RK]-x-[RK] and[RK]-V-x-[FW]/[RK]-x-V-x-[FW](A)[RK]-V-x-F/[RK]-x-V-x-F(B) motifs.

PREFERRED EMBODIMENTS OF THE INVENTION

The following terms that are used throughout the remaining specificationand claims and should be understood to mean, besides the genericdefinition, the following more precise definition; it being understoodthat the generic definitions are also included.

As used herein, the word “aspect” means any technical feature or elementof the claimed invention.

As used herein, the word “mimick” means close resemblance to either instructure and/ or function.

As used herein the word “isolated” means taken from the naturalenvironment. Isolated does not necessarily mean that what is taken fromthe natural environment is 100% purified.

As used herein the term “biochemical test” means any test which iscapable of identifying an interacting polypeptide or protein. Examplesof a biochemical tests include, but are not limited to,immunoprecipitation, use of antibodies, either monoclonal or polyclonal,oligonucleotide probes that can be labelled with radioactivity or anenzyme, GST pulldown (gel filtration experiment revealing size ofmacromolecular complexes) as described in Ayllón et al EMBO J. 192237-2246 (2000), and the like.

As used herein, the term “motif” means a particular amino acid sequenceor sequences which are similar and have the same function in differentcellular environments. A motif has some fixed amino acids and somevariable ones.

As used herein and throughout the entire specification and in theclaims, when several amino acids are bracketed, this means that theamino acid within the brackets can be one or the other. Thus [RK] meansthat the motif can have either R or K.

As used herein, the word “inhibits” means to prevent the specificinteractions, regardless of the mechanism of this prevention.

As used herein a “pharmaceutical composition” includes, but is notlimited to, the peptides of the present invention disclosed throughoutthe specification and a pharmaceutically acceptable carrier. Thispharmaceutical composition comprises a pharmaceutically acceptableamount of the peptides of the present invention. The pharmaceuticallyacceptable amount can be estimated from cell culture assays. Forexample, a dose can be formulated in animal models to achieve acirculating concentration range that includes or encompasses aconcentration point or range having the desired effect in an in vitrosystem. This information can thus be used to accurately determine thedoses in animals, including humans.

The therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms in a patient. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or in experimental animals.For example, the LD50 (the dose lethal to 50% of the population) as wellas the ED50 (the dose therapeutically effective in 50% of thepopulation) can be determined using methods known in the art. The doseratio between toxic and therapeutic effects is the therapeutic indexwhich can be expressed as the ratio between LD 50 and ED50 compoundsthat exhibit high therapeutic indexes.

The data obtained from the cell culture and animal studies can be usedin formulating a range of dosage of such compounds which lies preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity.

The pharmaceutical composition can be administered via any route such aslocally, orally, systemically, intravenously, intramuscularly,mucosally, using a patch and can be encapsulated in liposomes,microparticles, microcapsules, and the like. The pharmaceuticalcomposition can be embedded in liposomes or even encapsulated. Thepharamaceutical composition can also be in a lyophilized form.

Any pharmaceutically acceptable carrier or adjuvant can be used in thepharmaceutical composition. The modulating compound will be for instancein a soluble form combined with a pharmaceutically acceptable carrier.The techniques for formulating and administering these compounds can befound in “Remington's Pharmaceutical Science” Mack Publication Co.,Easton, Pa., latest edition.

The mode of administration optimum dosages and galenic forms can bedetermined by the criteria known in the art taken into account theseriousness of the general condition of the mammal, including the human,the tolerance of the treatment and the side effects.

“Pharmaceutical compositions” also include vectors which can beadministered directly in vivo or can be combined with specific cellsthat can be or may not be extracted from the animal to be treated. Typesof cells include all eukaryotic and prokaryotic cells including muscle,heart, liver, lung, brain cells, thymocytes, blood and the like. Plantand bacterial cells are also encompassed in the present invention sincethe PP1 is found in many different types of plant and bacterial cells.Yeast cells are also encompassed by the present invention. In fact anycell that contains PP1 is encompassed by the present invention.

Thus encompassed by the term “pharmaceutical compositions” are includedall forms of not only classic pharmaceutical administration, but alsoinclude gene therapy.

By the term “support” is meant any type of object on which peptides canbe immobilized. The type of support includes, but is not limited to,costar wells, beads, resins, glass chips, membranes and the like. Anysupport which can be used to immobilize proteins or peptides can be usedin the methods of the present invention.

The term “animal” encompasses any living being which is not vegetal,including vertebrates such as mammals, birds, reptiles, amphibians andfish.

As used herein the terms “polynucleotides”, “nucleic acids” and“oligonucleotides” are used interchangeably and include, but are notlimited to RNA, DNA, RNA/DNA sequences of more than one nucleotide ineither single chain or duplex form. The polynucleotide sequences of thepresent invention may be prepared from any known method including, butnot limited to, any synthetic method, any recombinant method, any exvivo generation method and the like, as well as combinations thereof.

Polynucleotides which can hybridize to any of the polynucleotidesdiscussed above are also covered by the present invention. Suchpolynucleotides are referred to herein as “hybridizing” polynucleotides.Hybridizing polynucleotides can be useful as probes or primers, forexample.

According to an embodiment of the present invention, such hybridizingmolecules are at least 10 nucleotides in length. According to anotherembodiment, they are at least 25 or at least 50 nucleotides in length.

In an embodiment, the hybridizing molecules will hybridize to suchmolecules under stringent hybridization conditions. One example ofstringent hybridization conditions is where attempted hybridization iscarried out at a temperature of from about 35° C. to about 65° C. usinga salt solution which is about 0.9 molar. However, the skilled personwill be able to vary such conditions as appropriate in order to takeinto account variables such as probe length, base composition, type ofions present, etc.

By “preventing or treating” is meant to manage a disease or medicalcondition or to arrest the onset of a disease or a medical condition.

More specifically, the present invention relates to a peptide or a setof peptides which mimicks both motifs M1 and M2 and which affects theBcl-x_(L)/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c interactions,wherein the motif M1 has the sequence FXX[RK]X[RK], and the motif M2 hasthe sequence [RK]VX[FW] or [RK]XVX[FW], wherein X is any amino acid.

In a specific embodiment, the present invention relates to set ofpeptides comprising the R (NWGRIVAFFSF) peptide and the F (GDEFELRYRRAF)peptide.

The present invention does not only encompass the specific peptides orset of peptides described above, but also modified peptides in which anamino acid is deleted, added or changed while retaining the samefunction of inhibiting the Bcl-x_(L)/PP1c, the Bcl-w/PP1c, or theBcl-2/Bad/PP1c interactions. In particular, chemical analogs of theamino acids can be used, such as phosphorylated or thiophosphorylatedamino acids. For example, it has been demonstrated that a phosphorylatedY is similar to the F residue. Thus, these two amino acids may beinterchanged without effecting the function of the motif.

Peptidic analogues of the motifs are thus encompassed in the presentinvention. These analogues include those structures that are similar infunction but are not identical in composition. An example of an analogueis a polypeptide that has chemically modified amino acids which arephosphorylated and cannot be dephosphorylated by the cellular enzymes.

The peptides or sets of peptides according to the present invention, orincluded in the compositions and kits of the invention, can encompasstwo or more motifs on the same molecule, or 2 to 5 motifs, or 2 to 10motifs, which motifs are described above, having a spacer between them.The spacer may be a sequence of amino acids or a hydrocarbon chaininterposed by covalent linkages. A spacer can also be any chemicalentity which can serve to connect the at least two motifs, and thespacer can also contain regulatory sequences between the at least twomotifs.

In another embodiment, the spacer of the present invention has fromabout 1 to about 50 amino acids. In a particular embodiment, the spacerhas about 36 amino acids.

In another embodiment, the present invention relates to a fusionpolypeptide or protein containing at least one motif of the presentinvention. This fusion polypeptide or protein can be made according tomethods known in the art such as those described in Sambrook et al,Molecular Cloning A Laboratory Manual 2^(nd) Edition (1989). Aparticular fusion polypeptide or protein of the invention comprises oneor several motifs M1 or M2, linked with at least one fusogenic peptidewhich will help the fusion polypeptide or protein enter target cells.Said fusogenic peptide can be for example a viral epitope or a ligand toa specific cell receptor such as chemokine receptors.

The above motifs, their analogues and modifications can be synthesizedusing, for example, an “Applied System” synthesizer or by Merrifieldtype solid phase synthesis (See, Merrifield, Adv. Enzmol. Related AreasMol. Biol. (1969) 32;221-96 or Merrfield, Recent Prog. Horm. Res.(1967); 23;451-82) The motifs can also be recombinantly produced.

The nucleic acid sequence encoding the motifs can be inserted into anexpression vector which contains the necessary elements for thetranscription and translation of the inserted peptide/protein-codingsequence. Such transcription elements include a regulatory region and apromoter. Thus, the nucleic acid which can encode a marker compound ofthe present invention is operably linked to a promoter in the expressionvector. The expression vector can also include a replication origin.

A wide variety of host/expression vector combinations are employed inexpressing the nucleic acids of the present invention. Useful expressionvectors that can be used include, for example, segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors include,but are not limited to, derivatives of SV40 and pcDNA and knownbacterial plasmids such as col EI, pCR1, pBR322, pMal-C2, pET, pGEX asdescribed by Smith et al (1988), pMB9 and derivatives thereof, plasmidssuch as RP4, phage DNAs such as the numerous derivatives of phage I suchas NM989, as well as other phage DNA such as M13 and filamentous singlestranded phage DNA; yeast plasmids such as the 2 micron plasmid orderivatives of the 2 m plasmid, as well as centomeric and integrativeyeast shuttle vectors; vectors useful in eukaryotic cells such asvectors useful in insect or mammalian cells; vectors derived fromcombinations of plasmids and phage DNAs, such as plasmids that have beenmodified to employ phage DNA or the expression control sequences; andthe like.

For example in a baculovirus expression system, both non-fusion transfervectors, such as, but not limited to pVL941 (BamHI cloning siteSummers), pVL1393 (BamHI, SmaI, XbaI, EcoRI, NotI, XmaIII, BgIII andPstI cloning sites; Invitrogen), pVL1392 (BgIII, PstI, NotI, XmaIII,EcoRI, XbalI, SmaI and BamHI cloning site; Summers and Invitrogen) andpBlueBacIII (BamHI, BglII, PstI, NcoI and HindIII cloning site, withblue/white recombinant screening, Invitrogen), and fusion transfervectors such as, but not limited to, pAc700 (BamHI and KpnI cloningsites, in which the BamHI recognition site begins with the initiationcodon; Summers), pAc701 and pAc70-2 (same as pAc700, with differentreading frames), pAc360 (BamHI cloning site 36 base pairs downstream ofa polyhedrin initiation codon; Invitrogen (1995)) and pBlueBacHisA, B, C(three different reading frames with BamHI, BglII, PstI, NcoI andHindIII cloning site, an N-terminal peptide for ProBond purification andblue/white recombinant screening of plaques; Invitrogen (220) can beused.

Mammalian expression vectors contemplated for use in the inventioninclude vectors with inducible promoters, such as the dihydrofolatereductase promoters, any expression vector with a DHFR expressioncassette or a DHFR/methotrexate co-amplification vector such as pED(PstI, SalI, SbaI, SmaI and EcoRI cloning sites, with the vectorexpressing both the cloned gene and DHFR; Kaufman, 1991). Alternativelya glutamine synthetase/methionine sulfoximine co-amplification vector,such as pEE14 (HindIII, XbalI, SmaI, SbaI, EcoRI and BclI cloning sitesin which the vector expresses glutamine synthetase and the cloned gene;Celltech). A vector that directs episomal expression under the controlof the Epstein Barr Virus (EBV) or nuclear antigen (EBNA) can be usedsuch as pREP4 (BamHI, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII andKpnI cloning sites, constitutive RSV-LTR promoter, hygromycin selectablemarker; Invitrogen), pCEP4 (BamHI, SfiI, XhoI, NotI, NheI, HindIII,NheI, PvuII and KpnI cloning sites, constitutive hCMV immediate earlygene promoter, hygromycin selectable marker; Invitrogen), pMEP4 (KpnI,PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamHI cloning sites, induciblemethallothionein IIa gene promoter, hygromycin selectable marker,Invitrogen), pREP8 (BamHI, XhoI, NofI, HindIII, NheI and KpnI cloningsites, RSV-LTR promoter, histidinol selectable marker; Invitrogen),pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI, BamHI cloning sites,RSV-LTR promoter, G418 selectable marker, Invitrogen), and pEBVHis(RSV-LTR promoter, hygromycin selectable marker, N-terminal peptidepurifiable via ProBond resin and cleaved by enterokinase; Invitrogen).

Selectable mammalian expression vectors for use in the inventioninclude, but are not limited to, pRc/CMV (HindIII, BstXI, NotI, SbaI andApaI cloning sites, G418 selection, Invitrogen), pRc/RSV (HindII, SpeI,BstXI, NotI, XbaI cloning sites, G418 selection, Invitrogen) and thelike. Vaccinia virus mammalian expression vectors (see, for exampleKaufman 1991 that can be used in the present invention include, but arenot limited to, pSC11 (SmaI cloning site, TK- and β-gal selection),pMJ601 (SalI, SmaI, AfiI, NarI, BspMII, BamHI, ApaI, NheI, SacII, KpnIand HindIII cloning sites; TK- and β-gal selection), pTKgptF1S (EcoRI,PstI, SalII, AccI, HindII, SbaI, BamHI and Hpa cloning sites, TK or XPRTselection) and the like.

Yeast expression systems that can also be used in the present include,but are not limited to, the non-fusion pYES2 vector (XbaI, SphI, ShoI,NotI, GstXI, EcoRI, BstXI, BamHI, SacI, KpnI and HindIII cloning sites,Invitrogen), the fusion pYESHisA, B, C (XbalI, SphI, ShoI, NotI, BstXI,EcoRI, BamHI, SacI, KpnI and HindIII cloning sites, N-terminal peptidepurified with ProBond resin and cleaved with enterokinase; Invitrogen),pRS vectors and the like.

Consequently, mammalian and typically human cells, as well as bacterial,yeast, fungi, insect, nematode and plant cells an used in the presentinvention and may be transfected by the nucleic acid or recombinantvector as defined herein.

Examples of suitable cells include, but are not limited to, VERO cells,HELA cells such as ATCC No. CCL2, CHO cell lines such as ATCC No. CCL61,COS cells such as COS-7 cells and ATCC No. CRL 1650 cells, W138, BHK,HepG2, 3T3 such as ATCC No. CRL6361, A549, PC12, K562 cells, 293 cells,Sf9 cells such as ATCC No. CRL1711, Cv1 cells such as ATCC No. CCL70 andJURKAT cells such as ATCC No. Tib152.

Other suitable cells that can be used in the present invention include,but are not limited to, prokaryotic host cells strains such asEscherichia coli, (e.g., strain DH5-α), Bacillus subtilis, Salmonellatyphimurium, or strains of the genera of Pseudomonas, Streptomyces andStaphylococcus, parasites like Apicomplexan parasites (Plasmodia,Toxoplasma, Cryptosporidia), Leishmania or Trypanosoma.

Further suitable cells that can be used in the present invention includeyeast cells such as those of Saccharomyces such as Saccharomycescerevisiae or Prombe.

The above-described motifs and peptides are involved. in bindingBcl-x_(L) and Bcl-w to PP1c and are thus targeting subunits involved incontrol of all PP1 binding. Thus, due to their implication in PP1binding, these motifs are important for the regulation of any diseaseconcerned with phosphatase regulation in all types of cells includingall eukaryotic and prokaryotic cells including muscle, heart, liver,lung, brain cells, thymocytes, blood and the like. Plant and bacterialcells are also encompassed in the present invention since the PP1 isfound in many different types of plant and bacterial cells. Yeast cellsare also encompassed by the present invention. In fact any cell thatcontains PP2 is encompassed by the present invention.

More importantly, this regulation can effect the downstream regulationof, for example, hepatic glycogen synthesis which in turn would lowerblood glucose levels and thus treat diabetes in which hyperglycemia is asevere problem. Thus, the present invention relates to treating diabetesby administering to an animal in need of such treatment apharmaceutically acceptable amount of the peptides of the presentinvention which are described in detail within.

In yet another embodiment, the present invention relates to theadministration of the peptides of the present invention to normalizehigh blood pressure. A decrease in phosphorylation of the PP1 complexactivates the smooth muscle myosin light chains and hence relaxes smoothmuscles (see, Uehata at al., 1997). Therefore, the present inventionalso relates to administering to an animal a pharmaceutically effectiveamount of the peptides of the present invention described within toprevent or treat hypertension.

in yet another embodiment the present invention relates to treatingneurological disorders by modulating neurological receptors and ionchannels. Therefore, the present invention also relates to treatingneurological disorders by administering to an animal in need of suchtreatment a pharmaceutically effective amount of the peptides of thepresent invention described within to prevent or treat the neurologicaldisorders, such as Parkinson's disease.

More specifically, the present invention relates to treating Alzheimer'sdisease. Phosphorylation is known to play a role in effecting theβ-amyloid precursor which causes the plagues in Alzheimer's disease.Thus, the present invention relates to treating Alzheimer's disease byadministering to an animal in need of such treatment a pharmaceuticallyeffective amount of the peptides of the present invention describedwithin to prevent or treat Alzheimer's disease.

In yet another embodiment, the present invention relates to treatingviral or microbial infections and more specifically herpes simplexvirus, Myocbacterium tuberculosis and AIDS by administering to an animalin need of such treatment a pharmaceutically effective amount of thepeptides of the present invention described within to prevent or treatthe viral infection.

The present invention is especially useful for treating AIDS since bothTAT and reverse transcriptase of the HIV-1 virus could be PP1 bindingproteins.

In yet another embodiment, the present invention relates to treatingparasitic infections such as malaria, theileria or cryptosporidium byadministering to an animal in need of such treatment a pharmaceuticallyeffective amount of the peptides of the present invention describedwithin to prevent or treat these parasitic infections.

The above-mentioned treatments involve administering to an animal inneed of such treatment a pharmaceutically acceptable amount of a peptideor a set of peptides which mimicks both motifs M1 and M2 and whichinhibits the Bcl-x_(L)/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1cinteractions. For example, a set of peptides comprising the R(NWGRIVAFFSF) peptide and the F (GDEFELRYRRAF) peptide, analogues ofthese peptides or their functional equivalents in a pharmaceuticallyacceptable vehicle can be administered to said animal.

In another embodiment the present invention relates to pharmaceuticalcompositions which comprises a peptide or a set of peptides whichmimicks both motifs M1 and M2 and which inhibits the Bcl-x_(L)/PP1c, theBcl-w/PP1c, or the Bcl-2/Bad/PP1c interactions. Such a composition canfor example comprise the R (NWGRIVAFFSF) peptide and the F(GDEFELRYRRAF) peptide, analogues of these peptides or their functionalequivalents in a pharmaceutically acceptable vehicle.

The pharmaceutically acceptable vehicle includes, but is not limited to,saline, adjuvants and the like, discussed more extensively above.

The present invention is not limited to solely administering thepeptides described within as a “neat” pharmaceutical composition, butalso as a pharmaceutical composition in gene therapy, using vectors thatencode polypeptides according to the present invention.

More specifically ex vivo and in vitro gene therapy is part of thepresent invention. In this respect, any of the methodologies relating togene therapy available within the art can be used in the practice of thepresent invention such as those described by Goldspiel et al Clin.Pharm. 12 pgs. 488-505 (1993).

Delivery of the therapeutic nucleic acid into a patient can be direct invivo gene therapy (i.e., the patient is directly exposed to the nucleicacid or nucleic acid-containing vector) or indirect ex vivo gene therapy(i.e., cells are first transformed with the nucleic acid in vitro andthen transplanted into the patient).

For example for in vivo gene therapy, an expression vector containingthe nucleic acid is administered in such a manner that it becomesintracellular, i.e., by infection using a defective or attenuatedretroviral or other viral vectors as described, for example in U.S. Pat.No. 4,980,286 or by Robbins et al, Pharmacol. Ther., 80 No. 1 pgs. 35-47(1998).

The various retroviral vectors that are known in the art are such asthose described in Miller at al. (Meth. Enzymol. 217 pgs. 581-599(1993)) which have been modified to delete those retroviral sequenceswhich are not required for packaging of the viral genome and subsequentintegration into host cell DNA. Also adenoviral vectors can be usedwhich are advantageous due to their ability to infect non-dividing cellsand such high-capacity adenoviral vectors are described in Kochanek(Human Gene Therapy, 10, pgs. 2451-2459 (1999)). Chimeric viral vectorsthat can be used are those described by Reynolds et al. (MolecularMedecine Today, pgs. 25-31 (1999)). Hybrid vectors can also be used andare described by Jacoby et al. (Gene Therapy, 4, pgs. 1282-1283 (1997)).

Direct injection of naked DNA or through the use of microparticlebombardment (e.g., Gene Gun®; Biolistic, Dupont) or by coating it withlipids can also be used in gene therapy. Cell-surfacereceptors/transfecting compounds or through encapsulation in liposomes,microparticles or microcapsules or by administering the nucleic acid inlinkage to a peptide which is known to enter the nucleus or byadministering it in linkage to a ligand predisposed to receptor-mediatedendocytosis (See Wu & Wu, J. Biol. Chem., 262 pgs. 4429-4432 (1987)) canbe used to target cell types which specifically express the receptors ofinterest.

In another embodiment a nucleic acid ligand compound can be produced inwhich the ligand comprises a fusogenic viral peptide designed so as todisrupt endosomes, thus allowing the nucleic acid to avoid subsequentlysosomal degradation. The nucleic acid can be targeted in vivo for cellspecific endocytosis and expression by targeting a specific receptorsuch as that described in WO92/06180, WO93/14188 and WO 93/20221.Alternatively the nucleic acid can be introduced intracellularly andincorporated within the host cell genome for expression by homologousrecombination (See Zijlstra et al, Nature, 342, pgs. 435-428 (1989)).

In ex vivo gene therapy, a gene is transferred into cells in vitro usingtissue culture and the cells are delivered to the patient by variousmethods such as injecting subcutaneously, application of the cells intoa skin graft and the intravenous injection of recombinant blood cellssuch as hematopoietic stem or progenitor cells.

Cells into which a nucleic acid can be introduced for the purposes ofgene therapy include, for example, epithelial cells, endothelial cells,keratinocytes, fibroblasts, muscle cells, hepatocytes and blood cells.The blood cells that can be used include, for example, T-lymphocytes,B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils,megakaryotcytes, granulocytes, hematopoietic cells or progenitor cellsand the like.

The polypeptides and complexes of polypeptides of the invention alsofind use in raising antibodies. Thus, the present invention providesantibodies, which can be monoclonal or polyclonal.

Thus, the polypeptides and complexes of the invention can be used as animmunogen to generate antibodies which specifically bind such animmunogen. Antibodies of the invention include, but are not limited topolyclonal, monoclonal, bispecific, humanized or chimeric antibodies,single chain antibodies, Fab fragments and F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. The term“antibody” as used herein refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bindsan antigen. The immunoglobulin molecules of the invention can be of anyclass (e.g., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulinmolecule.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., ELISA(enzyme-linked immunosorbent assay). For example, to select antibodieswhich recognize a specific domain of a polypeptide of the invention(e.g., a Selected Interacting Domain), one can assay generatedhybridomas for a product which binds to a polypeptide fragmentcontaining such domain. For selection of an antibody that specificallybinds a first polypeptide homolog but which does not specifically bindto (or binds less avidly to) a second polypeptide homolog, one canselect on the basis of positive binding to the first polypeptide homologand a lack of binding to (or reduced binding to) the second polypeptidehomolog.

For preparation of monoclonal antibodies (mAbs) directed toward apolypeptide of the invention or a fragment or an analog thereof, anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture may be used. For example, the hybridomatechnique originally developed by Kohler and Milstein (1975, Nature256:495-497), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAbs of the invention can be cultivated in vitroor in vivo. In an additional embodiment of the invention, monoclonalantibodies can be produced in germ-free animals utilizing knowntechnology (PCT/US90/02545, incorporated herein by reference).

The monoclonal antibodies include but are not limited to humanmonoclonal antibodies and chimeric monoclonal antibodies (e.g.,human-mouse chimeras). A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a human immunoglobulin constant region and a variableregion derived from a murine mAb. (See, e.g., Cabiliy et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.)

Chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in PCT Publication No. WO 87/02671; European. PatentApplication 184,187; European Patent Application 171,496; EuropeanPatent Application 173,494; PCT Publication No. WO 86/01533; U.S. Pat.No. 4,816,567; European Patent Application 125,023; Better et at, 1988,Science 240:1041-1043; Liu et at, 1987, Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et at, 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et at, 1987,Canc. Res. 47:999-1005; Wood et at, 1985, Nature 314:446-449; and Shawet at, 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science229:1202-1207; Oi et al., 1986, Bio/Techniques 4:214; U.S. Pat. No.5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al,(1988) Science 239:1534; and Beidler et at, 1988, J. Immunol.141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chain genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of a BPIof the invention. Monoclonal antibodies directed against the antigen canbe obtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.) andGenpharm (San Jose, Calif.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al. (1994) Bio/technology12:899-903).

The antibodies of the present invention can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.More specifically, such phage can be utilized to display antigen bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Phage display methods that can be used to make the antibodies of thepresent invention include those disclosed in Brinkman et al., J.Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958(1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances inImmunology 57:191-280 (1994); PCT Application No. PCT/GB91/01134; PCTPublications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and5,969,108; each of which is incorporated herein by reference in itsentirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988).

The invention further provides for the use of bispecific antibodies,which can be made by methods known in the art. Traditional production offull length bispecific antibodies is based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Milstein et al., 1983, Nature 305:537-539).Because of the random assortment of immunoglobulin heavy and lightchains, these hybridomas (quadromas) produce a potential mixture of 10different antibody molecules, of which only one has the correctbispecific structure. Purification of the correct molecule, which isusually done by affinity chromatography steps, is rather cumbersome, andthe product yields are low. Similar procedures are disclosed in WO93/08829, published 13 May 1993, and in Traunecker et al., 1991, EMBO J.10:3655-3659.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion can bewith an immunoglobulin heavy chain constant domain, comprising at leastpart of the hinge, CH2, and CH3 regions. Generally, the firstheavy-chain constant region (CH1) containing the site necessary forlight chain binding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In another embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690 published Mar. 3,1994. For further details for generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology, 1986, 121:210.

The invention provides functionally active fragments, derivatives oranalogs of the anti-polypeptide immunoglobulin molecules. Functionallyactive means that the fragment, derivative or analog is able to elicitanti-anti-idiotype antibodies (i.e., tertiary antibodies) that recognizethe same antigen that is recognized by the antibody from which thefragment, derivative or analog is derived. Specifically, in anembodiment, the antigenicity of the idiotype of the immunoglobulinmolecule can be enhanced by deletion of framework and CDR sequences thatare C-terminal to the CDR sequence that specifically recognizes theantigen. To determine which CDR sequences bind the antigen, syntheticpeptides containing the CDR sequences can be used in binding assays withthe antigen by any binding assay method known in the art.

The present invention provides antibody fragments such as, but notlimited to, F(ab′)2 fragments and Fab fragments. Antibody fragmentswhich recognize specific epitopes can be generated by known techniques.F(ab′)2 fragments consist of the variable region, the light chainconstant region and the CH1 domain of the heavy chain and are generatedby pepsin digestion of the antibody molecule. Fab fragments aregenerated by reducing the disulfide bridges of the F(ab′)2 fragments.The invention also provides heavy chain and light chain dimmers of theantibodies of the invention, or any minimal fragment thereof such as Fvsor single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature334:544-54), or any other molecule with the same specificity as theantibody of the invention. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli can be used (Skerra etal., 1988, Science 242:1038-1041).

In other embodiments, the invention provides fusion proteins of theimmunoglobulins of the invention (or functionally active fragmentsthereof), for example in which the immunoglobulin is fused via acovalent bond (e.g., a peptide bond), at either the N-terminus or theC-terminus to an amino acid sequence of another protein (or portionthereof, preferably at least 10, 20 or 50 amino acid portion of theprotein) that is not the immunoglobulin. In an embodiment, theimmunoglobulin, or a fragment thereof, is covalently linked to the otherprotein at the N-terminus of the constant domain. As stated above, suchfusion proteins may facilitate purification, increase half-life in vivo,and enhance the delivery of an antigen across an epithelial barrier tothe immune system.

The immunoglobulins of the invention include analogs and derivativesthat are either modified, i.e., by the covalent attachment of any typeof molecule as long as such covalent attachment that does not impairimmunospecific binding. For example, but not by way of limitation, thederivatives and analogs of the immunoglobulins include those that havebeen further modified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, etc. Additionally, the analog orderivative can contain one or more non-classical amino acids.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the polypeptides of theinvention, e.g., for imaging or radioimaging these proteins, measuringlevels thereof in appropriate physiological samples, in diagnosticmethods, etc. and for radiotherapy.

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, for instance, by chemicalsynthesis or by recombinant expression, and are preferably produced byrecombinant expression technique.

Recombinant expression of antibodies, or fragments, derivatives oranalogs thereof, requires construction of a nucleic acid that encodesthe antibody. If the nucleotide sequence of the antibody is known, anucleic acid encoding the antibody can be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,1994, BioTechniques 17:242), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding antibody, annealing and ligation of those oligonucleotides, andthen amplification of the ligated oligonucleotides by PCR.

Alternatively, the nucleic acid encoding the antibody can be obtained bycloning the antibody. If a clone containing the nucleic acid encodingthe particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody can beobtained from a suitable source (e.g., an antibody cDNA library, or cDNAlibrary generated from any tissue or cells expressing the antibody) byPCR amplification using synthetic primers hybridizable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence.

If an antibody molecule that specifically recognizes a particularantigen is not available (or a source for a cDNA library for cloning anucleic acid encoding such an antibody), antibodies specific for aparticular antigen can be generated by any method known in the art, forexample, by immunizing an animal, such as, a rabbit, to generatepolyclonal antibodies or, by generating monoclonal antibodies.Alternatively, a clone encoding at least the Fab portion of the antibodymay be obtained by screening Fab expression libraries (e.g., asdescribed in Huse et al., 1989, Science 246:1275-1281) for clones of Fabfragments that bind the specific antigen or by screening antibodylibraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane etal., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid encoding at least the variable domain of theantibody molecule is obtained, it can be introduced into a vectorcontaining the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g., PCT Publication WO 86/05807; PCTPublication WO 89/01036; and U.S. Pat. No. 5,122,464). Vectorscontaining the complete light or heavy chain for co-expression with thenucleic acid to allow the expression of a complete antibody molecule arealso available. Then, the nucleic acid encoding the antibody can be usedto introduce the nucleotide substitution(s) or deletion(s) necessary tosubstitute (or delete) the one or more variable region cysteine residuesparticipating in an intrachain disulfide bond with an amino acid residuethat does not contain a sulfhydyl group. Such modifications can becarried out by any method known in the art for the introduction ofspecific mutations or deletions in a nucleotide sequence, for example,but not limited to, chemical mutagenesis, in vitro site directedmutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551), PCTbased methods, etc.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human antibodyconstant region, e.g., humanized antibodies.

Once a nucleic acid encoding an antibody molecule of the invention hasbeen obtained, the vector for the production of the antibody moleculecan be produced by recombinant DNA technology using techniques wellknown in the art. Thus, methods for preparing the protein of theinvention by expressing nucleic acid containing the antibody moleculesequences are described herein. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining an antibody molecule coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro. recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al. (1990, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.) and Ausubel et al. (eds., 1998, Current Protocols inMolecular Biology, John Wiley & Sons, NY).

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention.

The host cells used to express a recombinant antibody of the inventioncan be either bacterial cells such as Escherichia coli, or eukaryoticcells, especially for the expression of whole recombinant antibodymolecule. More specifically, mammalian cells such as Chinese hamsterovary cells (CHO), in conjunction with a vector such as the majorintermediate early gene promoter element from human cytomegalovirus isan effective expression system for antibodies (Foecking et al., 198,Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).

A variety of host-expression vector systems can be utilized to expressan antibody molecule of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest can beproduced and subsequently purified, but also represent cells which can,when transformed or transfected with the appropriate nucleotide codingsequences, express the antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter, the vaccinia virus 7.5Kpromoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions comprising an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coil expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence can be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors can also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence can be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). In mammalian host cells, a number ofviral-based expression systems (e.g., an adenovirus expression system)can be utilized.

As discussed above, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein.

For long-term, high-yield production of recombinant antibodies, stableexpression is useful. For example, cells lines that stably express anantibody of interest can be produced by transfecting the cells with anexpression vector comprising the nucleotide sequence of the antibody andthe nucleotide sequence of a selectable (e.g., neomycin or hygromycin),and selecting for expression of the selectable marker. Such engineeredcell lines can be useful in screening and evaluation of compounds thatinteract directly or indirectly with the antibody molecule.

The expression levels of the antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel. Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et at, 1983, Mol. Cell. Biol.3:257).

The host cell can be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors can contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector can be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once the antibody molecule of the invention has been recombinantlyexpressed, it can be purified by any method known in the art forpurification of an antibody molecule, for example, by chromatography(e.g., ion exchange chromatography, affinity chromatography such as withprotein A or specific antigen, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins.

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897).In this system, the gene of interest is subcloned into a vacciniarecombination plasmid such that the open reading frame of the gene istranslationally fused to an amino-terminal tag consisting of sixhistidine residues. The tag serves as a matrix binding domain for thefusion protein. Extracts from cells infected with recombinant vacciniavirus are loaded onto Ni2⁺ nitriloacetic acid-agarose columns andhistidine-tagged proteins are selectively eluted withimidazole-containing buffers.

In another embodiment, antibodies of the invention or fragments thereofare conjugated to a diagnostic or therapeutic moiety. The antibodies canbe used for diagnosis or to determine the efficacy of a given treatmentregimen. Detection can be facilitated by coupling the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive nuclides, positronemitting metals (for use in positron emission tomography), andnonradioactive paramagnetic metal ions. See generally U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics according to the present invention. Suitable enzymesinclude horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; suitable prosthetic groupsinclude streptavidin, avidin and biotin; suitable fluorescent materialsinclude umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride andphycoerythrin; suitable luminescent materials include luminol; suitablebioluminescent materials include luciferase, luciferin, and aequorin;and suitable radioactive nuclides include ¹²⁵I, ¹³¹I, ¹¹¹I and ⁹⁹Tc.

Antibodies of the invention or fragments thereof can be conjugated to atherapeutic agent or drug moiety to modify a given biological response.The therapeutic agent or drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietycan be a protein or polypeptide possessing a desired biologicalactivity. Such proteins can include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, α-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator, athrombotic agent or an anti-angiogenic agent, e.g., angiostatin orendostatin; or, a biological response modifier such as a lymphokine,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), nerve growth factor (NGF) or othergrowth factor.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al, “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (ads.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera at al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

An antibody with or without a therapeutic moiety conjugated to it can beused as a therapeutic that is administered alone or in combination withcytotoxic factor(s) and/or cytokine(s).

The present invention not only relates to the peptides, their use andadministration in pharmaceutically acceptable vehicles, but also tomethods of using the peptides and the motifs as disclosed within.

More specifically, the present invention has utilization for severalmethods in which the peptides described within are used for thefollowing purposes:

-   -   (1) A method of identifying a PP1-interacting polypeptide or        protein, comprising detecting in the sequence of said        polypeptide or protein, the presence of two PP1-binding motifs        M1 and M2.    -   (2) A method of screening compounds that interacts with PP1        regulators, comprising:    -   (a) immobilizing peptides described herein on a support; and    -   (b) testing the interaction of said compounds with said        immobilized peptides.    -   (3) A method of screening compounds that interact with PP1,        comprising:    -   (a) obtaining antibodies to peptides within the description of        the present invention; and    -   (b) testing the interaction of said compounds with said        antibodies.    -   (4) A method for testing molecules that inhibit or enhance the        PP1 activity or change its localization by interaction, said        method comprising:    -   (a) immobilizing peptides described within on a support; and    -   (b) testing the interaction of said compounds with said        immobilized peptides.

Also encompassed by the present invention are drugs that after using themethods of the present invention are found to inhibit and/or enhance PP1activity, PP1c activity or localization by interaction.

Besides methods, the present invention relates to kits. The kitscomprise, but are not limited to, peptides or sets of peptides whichmimick both the motifs M1 and M2 and which are able to inhibit theBcl-x_(L)/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c interactions,wherein the motif M1 has the sequence FXX[RK]X[RK], and the motif M2 hasthe sequence [RK]VX[FW] or [RK]XVX[FW], wherein X is any amino acid.Such kits can, for example, comprise the peptides R (NWGRIVAFFSF) and F(GDEFELRYRRAF). These peptides may be immobilized on any solid support,described above, and which includes resins, microstar wells, glass chipsand the like.

In yet another embodiment the present invention relates to a kitcontaining antibodies to peptides which mimick both the motifs M1 and M2and which are able to inhibit the Bcl-x_(L)/PP1c, the Bcl-w/PP1c, or theBc12/Bad/PP1c interactions. These antibodies can have been raisedagainst the peptides R (NWGRIVAFFSF) and F (GDEFELRYRRAF) peptides, theanalogues of these peptides or their functional equivalents.

Besides the peptides or the antibodies, the kits of the presentinvention also include reagents for interpreting the interaction. Suchreagents are known in the art and described above and in Sambrook(supra).

In yet another embodiment, the present invention concerns a method ofscreening cells to find PP1 interactions in the cells, said methodcomprising either using antibodies against the motifs M1 and M2 orpeptides including these motifs or their functional equivalents.

The present invention thus also relates to a new phosphatase-deriveddrug therapy based on the intracellular delivery of peptides with asequence or sequences surrounding the PP1/PP2 binding sites (M1 and M2motifs) identified in some interacting proteins. This drug therapyapproach, derived from chemical genetics (Gura, 2000), wouldspecifically inhibit the interaction of some medically important targetproteins with PP1/PP2A.

This therapeutic strategy, called “Peptidic knockout” of PP1/PP2pathways has the basis in two recent results. The first result was theidentification of a putative PP1 signature. Thus, from thecharacterization of two distinct PP1 binding motifs in Bcl-2 proteinsand their existence in most PP1 binding proteins, it can be concludedthat the combinatory presence of the motifs can be used as a predictivesignature for PP1 binding. Thus, the web site called “PP1 signature” (inpreparation at the Institut Pasteur) that contains all putative PP1sequences derived from a Swisprot library can be used to identifyputative PP1-binding targets of interest.

The second result was the identification of PP2A binding sites in fiveproteins. The PP2A binding sites of a viral encoded (HIV-1) Vpr and aparasitic Ck2a encoded by Theileria were recently mapped. From the dataobtained it can be said that the intracellular delivery of some peptidesmimicking these PP2A-binding sequences lead to apoptosis in tumors(Hela, Jurkat, S) or infected cells (Vpr or HIV-1 or Theileria).

Examples

The following examples can be performed using the materials and methodsdescribed below:

1. Materials and Methods 1.1. Cells and Culture

Ts1αβ is a murine T cell line expressing the α and β chains of the IL-2receptor (Pitton at al, 1993) that can be propagated independently inIL-2, IL-4 or IL-9. Cells were cultured in RPMI-1640 supplemented with5% heat-inactivated fetal calf serum 2 mM glutamine, 10 mM Hepes, 0.55mM arginine, 0.24 mM asparagine, 50 μLM 2-ME and 60 U/ml of IL-4.

1.2. Lymphokines, Antibodies, Reagents and Plasmids

Murine rlL-4 or supernatant of a HeLa subline transfected withpKCRIL-4.neo was used as a source of murine IL-4. Anti-Bcl-x_(L) andanti-Bcl-w antibody were from Calbiochem (La Jolla, Calif.),Transduction Laboratories (Lexington, Ky.) or StressGen Biotechnology(Victoria, Canada). Specific anti-PP1c was from UBI (Lake Placid, N.Y.),Calbiochem or Transduction Laboratories. Anti-Histones antibody was fromChemicon International (Temecula, Calif.). Anti-14-3-3 protein antibodywas from UBI (Lake Placid, N.Y.). Anti-Bad serine 112 and 136 were fromNew England BioLabs (Beverly, Mass.) and serine 155 was from CellSignaling Technology (Beverly, Mass.). Anti-Raf antibody was fromTransduction Laboratories. Anti-Pser and pan-Ras antibody were fromCalbiochem. Recombinant PP1c protein was from Calbiochem. Mito 2813(anti-mitochondrial pyruvate dehydrogenase) was provided by Dr Serrano,Centro Nacional de Biotecnologia (Madrid).

1.3. Immunoprecipitation and Western Blot

Cells (1×10⁷) were IL-4-stimulated or -deprived and lysed for 20 min at4° C. in lysis buffer (50 mM Tris HCl pH 8, 1% NP-40, 137 mM NaCl, 1 mMMgCl₂, 1 mM CaCl₂, 10% glycerol and protease inhibitors cocktail).Digitonin or detergent free buffers were also used forimmunoprecipitation. For phosphorylation analysis, the buffer was alsosupplemented with phosphatase inhibitors cocktail. Lysates wereimmunoprecipitated with the Appropriate antibody and Protein A Sepharosewas added. Alternatively, cells were lysed in Laemmli sample buffer andprotein extracts separated by SDS-PAGE, transferred to nitrocellulose,blocked and incubated with primary antibody. Membrane was washed andincubated with PO-conjugated secondary antibody. Proteins were developedusing the ECL.

1.4. In Vitro Phosphatase Assay

IL-4-stimulated cells (1×10⁷) were lysed in lysis buffer, supernatantswere immunoprecipitated with the corresponding antibody, followed byincubation with Protein A Sepharose. Immunoprecipitates were washed withphosphatase buffer (50 mM Tris HCl, pH 7.5, 0.1% 2-ME, 0.1 mM EDTA and 1mg/ml BSA) and mixed with [³²P] phosphorylase a, diluted in phosphatasebuffer supplemented with caffeine. The reaction was incubated (40 min at30° C.), stopped with 200 μl 20% TCA and centrifuged. A total of 185 μlof the supernatant were used to estimate the generation of freephosphate liberated from [³²P]phosphorylase a.

1.5. Peptide Synthesis

Peptides comprising the R/K X V/I X F (R) or F X X R X R (F) motif ofBcl-w and Bcl-x_(L), as well as the mutated peptides (see FIG. 8A forsequence) were prepared by automated spot synthesis into anaminoderivatized cellulose membrane. Membrane was blocked, incubatedwith purified PP1c and, after several washing steps, incubated withanti-PP1c antibody, followed by PO-conjugated secondary antibody. Spotswere developed using the ECL system.

R (NWGRIVAFFSF), F (GDEFELRYRRAF) or R* (NWGRIAAAFSF) peptides weresynthetized on an automated multiple peptide synthesizer using thesolid-phase procedure and standard Fmoc chemistry. The purity andcomposition of the peptides was confirmed by reverse-phase highperformance liquid chromatography and by amino acid analysis.

1.6. Protein-Protein Interaction Competition

The interaction Bcl-w/PP1c and Bcl-x_(L)/PP1c was competed by the R, F,or R* peptides. Lysates from IL-4-stimulated cells wereimmunoprecipitated with anti-Bad antibody and Protein A Sepharose wasadded. The interaction Bcl-w/PP1c and Bcl-x_(L)/PP1c was competed byincubation with R, F or R* peptides (30 min, room temperature). Afterwashing, immunoprecipitates were either assayed for protein phosphataseactivity or transferred to nitrocellulose and blotted with thecorresponding antibody.

1.7. Sense and Antisense Oligonucleotides

The phosphothioate analogous of the oligonucleotides from Bcl-x_(L) andBcl-w, including the ATG initiation codon were purchased from IsogenBioscience. The sequence of the sense and antisense oligonucleotides areas follow. Sense Bcl-x_(L), ATG TCT CAG AGC AAC; antisense Bcl-x_(L),GTT GCT CTG AGA CAT; sense Bcl-w, ATG GCG ACC CCA GCC; antisense Bcl-w,GGC TGG GGT CGC CAT.

2. Experimental Results

2.1. Identification of Bcl-w and Bcl-x_(L) as PP1c-Interacting Proteins

It was previously shown that anti-apoptotic molecule Bcl-2 is atargeting subunit of the serine/threonine phosphatase PP1c inIL-2-stimulated TS1αβ cells and that the sequence of Bcl-2 interactingwith PP1c is the R/K X V/I X F motif (Ayllón et al, 2001). Given thatBcl-x_(L) and Bcl-w also contain the well conserved R/K X V/I X F motifobserved in Bcl-2, the possibility that anti-apoptotic moleculesBcl-x_(L) and Bcl-w may be as well associated to PP1c in IL-4-stimulatedTS1αβ cells, which do not express Bcl-2 and express Bcl-x_(L) and Bcl-wwas explored. Reciprocal co-immunoprecipitation experiments ofcytoplasmic proteins under IL-4-stimulation or -deprivation conditionsusing specific antibodies was performed. PP1c and Bad were detected byWestern blot in anti-Bcl-x_(L) immunoprecipitates of IL-4 stimulatedcells, decreasing throughout the starvation period (FIG. 1A). Probingthe membrane with anti-Bcl-x_(L) antibody showed similar levels in allconditions analyzed. PP1c and Bad were also detected in anti-Bcl-wimmunoprecipitates of IL-4-stimulated cells, diminishing afterlymphokine deprivation (FIG. 1A). Membrane was also probed withanti-Bcl-w antibody, showing similar levels. Immunoprecipitation forcytoplasmic lysates with an irrelevant antibody, anti-p55 IL-2R chain,was not able to detect those associations (FIG. 1B). Similarly,Bcl-x_(L), Bcl-w and PP1c were detected in Bad immunoprecipitates andthe interaction among these proteins was also observed byimmunoprecipitation of detergent-free lysates as well as in cytoplasmicproteins isolated by digitonin lysis (data not shown). Theseassociations were also observed in freshly isolated thymocytes (FIG.1C). Given that the number of Bcl-x_(L)/PP1c/Bad and Bcl-w/PP1c/Badcomplexes decreases after IL-4-deprivation, down-regulation of theexpression of any of the proteins involved in the formation of thecomplex was analyzed. Thus, the total expression of Bcl-x_(L), Bcl-w,PP1c and Bad in IL-4-stimulated or -deprived TS1αβ cells was analyzed.All analyzed proteins were expressed in IL-4-stimulated or -deprivedcells (FIG. 2). As an internal control of protein loading, membraneswere probed with anti-Histones antibody. As the number of aggregatesdecreases after IL-4-deprivation without modification of totalexpression of the proteins of the complex, post-translationalmodifications of Bcl-x_(L) or Bcl-w may affect the formation of thetrimolecular complex was then analyzed. The status of serinephosphorylation of Bcl-x_(L) and Bcl-w was next analyzed. Cytoplasmicextracts from IL-4-stimulated or -deprived cells were immunoprecipitatedwith anti-Bcl-x_(L) or anti-Bcl-w and blotted with anti-Pser, anti-PP1c,anti-Bad, anti-Bcl-w and anti-Bcl-x_(L) specific antibodies (FIG. 3).Serine phosphorylation of. Bcl-x_(L) and Bcl-w was observed in controlIL-4-stimulated cells, decreasing after IL-4 deprivation. In agreementwith results in FIG. 1, the level of Bad and PP1c associated toBcl-x_(L) and Bcl-w diminishes upon lymphokine deprivation. This resultsuggests a correlation between serine phosphorylation of Bcl-x_(L) andBcl-w and formation of trimolecular complexes.

It was recently shown that IL-2, as well as IL-3, induces serinephosphorylation of Bad (Ayllón et al, 2000). FIG. 4A shows that IL-4induces Bad phosphorylation at serine 136 but not at serines 112 and155. Moreover, IL-4-deprivation induces serine 136 dephosphorylation ofBad. IL-2 stimulated cells (C, ser 112 and 136 phosphorylation) or COS(C, ser 155 phosphorylation) cells overexpressing Bad were used as apositive controls. IL-3-induced serine phosphorylation of Bad results inits association to the 14-3-3 protein, abolishing interaction with Bcl-x(Zhou et al, 2000). FIG. 4B shows that serine phosphorylation of Bad inresponse to IL-4 does not result in binding to 14-3-3 protein. Thisprotein was detected in total extracts from control IL-4 stimulatedcells (lane T) and was not observed neither in PP1c, nor in Bcl-x_(L) orBad immunoprecipitates of IL-4-stimulated or -deprived cells. As aninternal control, the interaction of Raf and the 14-3-3 protein in Rafimmunoprecipitates is shown (FIG. 4B).

FIG. 5A shows phosphatase activity in Bcl-x_(L), Bcl-w and Badimmunoprecipitates of IL-4-stimulated cells. The enzymatic activity inthe immunoprecipitates was measured using ³²P-labeled phosphorylase a assubstrate. It is interesting to notice that phosphatase activitydetected in Bcl-x_(L) and Bcl-w immunoprecipitates nearly corresponds tothe phosphatase activity observed in Bad immunoprecipitates. To confirmthat this phosphatase activity was due to PP1c, enzymatic activity wasestimated in Bad or Bcl-w immunoprecipitates from IL-4-stimulated cellsin the presence of different okadaic acid (OA) concentrations (FIG. 5B).OA concentrations that inhibit type 2A activity (10⁻⁹M) had no effect onBad- or Bcl-w-associated phosphatase activity in vitro. Addition of10⁻⁸M OA to Bcl-w or Bad immunoprecipitates results in ˜50% inhibitionof phosphatase activity, which is strongly reduced after addition of10⁻⁶M OA (FIG. 5B). The effect of OA on phosphatase activity was alsoestimated in supernatants of Bad and Bcl-w immunoprecipitates. OAconcentrations that had no effect on enzymatic activity in Bad and Bcl-wimmunoprecipitates (10⁻⁹M) shows ˜50% inhibition in the supematant, asexpected from an association of type 1 and type 2A activities (FIG. 5B).The selective effect of OA suggests that the phosphatase activityobserved in Bad and Bcl-w immunoprecipitates is PP1c.

2.2. Bcl-w and Bcl-x_(L) are New Targeting Subunits of PP1c

It was recently shown that Bcl-2 is a targeting subunit of PP1c (Ayllónet al, 2001). Given that Bcl-w and Bcl-x_(L) are also associated to PP1cand that sequence of binding site of Bcl-2 to PP1c is conserved inBcl-x_(L) and Bcl-w, it was hypothesized that these anti-apoptoticmolecules may be new targeting subunits of PP1c. To test thishypothesis, Bcl-x_(L) and Bcl-w was depleted by sequentialanti-Bcl-x_(L)-Bcl-w immunoprecipitation of cytoplasmic extracts of IL-4stimulated cells. Supernatant from the fourth anti-Bcl-x_(L)+Bcl-wimmunoprecipitation was immunoprecipitated with anti-Bad antibody (5th)and phosphatase activity estimated (FIG. 6A). Traces of Bad-associatedphosphatase activity were detected in Bcl-x_(L) and Bcl-w depletedextracts compared to the high level of activity observed in controlanti-Bad immunoprecipitates of IL-4-stimulated cells. Given that in theabsence of Bcl-x_(L) and Bcl-w significant Bad-associated phosphataseactivity was not detected, the possibility that these anti-apoptoticmolecules may control targeting of PP1c to Bad was explored. For thispurpose, anti-Bad immunoprecipitations were made in cytoplasmic extractsof IL-4 stimulated cells or in extracts depleted of Bcl-x_(L) and Bcl-w(5th). PP1c, Bcl-x_(L) and Bcl-w were detected in control anti-Badimmunoprecipitates and were not observed in anti-Bad immunoprecipitatesfrom extracts depleted of Bcl-x_(L) and Bcl-w (FIG. 6B). In a reciprocalexperiment, Bad, Bcl-x_(L) and Bcl-w were detected in anti-PP1cimmunoprecipitates of control cells and were not observed in PP1cimmunoprecipitates from extracts depleted of Bcl-x_(L) and Bcl-w (FIG.6B). This result suggests that Bcl-x_(L) and Bcl-w are needed forassociation of PP1c to Bad.

2.3. Determination of Bcl-x_(L) and Bcl-w Binding Site to PP1c

It has been described that R/K X V/I X F motif is shared by most of thePP1c targeting subunits (21, 23). It was shown that PP1c targetingsubunit Bcl-2 also shares this conserved motif (Ayllón et al, 2001).Interestingly, Bcl-x_(L) and Bcl-w sequences also contain this motif(FIG. 7B). To analyze whether this sequence of Bcl-x_(L) and Bcl-w wasinvolved in binding to PP1c, we generated nitrocellulose-immobilizedpeptides of Bcl-x_(L) and Bcl-w protein containing this motif. Membranewas incubated with purified PP1c protein. FIG. 7B shows the sequencesinteracting with PP1c. The R/K X V/I X F motif, present in Bcl-x_(L) andBcl-w, interact with PP1c and its mutation in critical V and F residuesstrongly reduces binding of Bcl-x_(L) and Bcl-w to PP1c (FIG. 7B).Analysis of the Bcl-2 binding sites to PP1c showed, in addition to R/K XV/I X F motif, two sequences (FSRRYR and FTARGR, that bind PP1c (Ayllónet al, 2001). Interestingly, similar sequences were as well observed inBcl-x_(L) and Bcl-w (FIG. 7A). FELRYR and FETRFR sequences of Bcl-x_(L)and Bcl-w respectively also interacts with PP1c and its mutation inhibitbinding to PP1c, although the affinity depends on the type of pointmutation (FIG. 7B). The interacting consensus F X X R X R motif wasdetermined by sequence comparison of Bcl-2, Bcl-x_(L) and Bcl-w.

To conclusively confirm that R/K X V/I X F and F X X R X R motifs areinvolved in binding of Bcl-x_(L) and Bcl-w to PP1c, competitionexperiments in trimolecular complexes were performed. Lysates fromIL-4-stimulated cells were immunoprecipitated with anti-Bad antibody andthe interaction Bcl-x_(L)/PP1c and Bcl-w/PP1c was competed using R*(NWGRIAAAFSF), R (NWGRIVAFFSF) or F (GDEFELRYRRAF) peptides (FIG. 8A).Bcl-x_(L), Bcl-w and PP1c were detected in control anti-Badimmunoprecipitates, as well as in anti-Bad immunoprecipitates treatedwith R* peptide. The amount of Bcl-x_(L), Bcl-w and PP1 c associated toBad decreases after competition with F or R peptide, being almostundetectable upon competition of Bad immunoprecipitates with F+Rpeptides (FIG. 8A). Similar level of Bad is observed in control orpeptide-treated anti-Bad immunoprecipitates. Finally, to confirm thatBcl-x_(L) and Bcl-w are targeting subunits of PP1c, we estimatedphosphatase activity in control or peptide-treated Badimmunoprecipitates. Phosphatase activity was detected in control or. R*peptide-treated immunoprecipitates (FIG. 8B), decreasing uponcompetition of the interaction with F or R peptides. Enzymatic activitywas strongly decreased upon competition with R and F peptides. As aninternal control, FIG. 8C shows the phosphatase activity in control orpeptide-treated Bad immunoprecipitates. The concentration of F or Rpeptide used was twice the concentration used in F+R peptide-treatedimmunoprecipitates. As in FIG. 8B, phosphatase activity was drasticallyreduced upon treatment of Bad immunoprecipitates with F+R peptides.Taken together, these results illustrate that Bcl-x_(L) and Bcl-w, aswell as Bcl-2, are PP1c targeting subunits.

2.4. Inhibition of PP1c Enzymatic Activity Blocks Apoptosis

As 1L-4-deprivation correlates with Bad dephosphorylation and apoptosis,it was hypothesized that inhibition of phosphatase activity by okadaicacid (OA) treatment may prevent Bad dephosphorylation and apoptosis.Treatment of the cells with 1 μM OA in the absence of IL-4 prevented Baddephosphorylation at serine 136 (FIG. 9A). No changes in total Badexpression were observed after OA treatment, suggesting that OA does notaffect protein expression. In addition, IL-4-deprived cells treated with1 μM OA for 6 h showed significant reduction in the fraction ofapoptotic cells compared with untreated cells (FIG. 9B). Finally,inhibition of Bcl-x_(L) and Bcl-w expression by antisenseoligonucleotide treatment of cells also induces apoptosis inIL-4-stimulated cells (FIG. 9C). The inhibition of Bcl-x_(L) and Bcl-wexpression upon antisense oligonucleotide treatment was estimated byWestern blot.

3. Bio-Infromatic Results-Predictive Signature for PP1 Interactions:Combinatorial Presence of [RK]VxF or [RK]xVxF and F-x-x-[RK]-x-[RK]Motifs in Characterized PP1 Binding Proteins

The combinatorial presence of these two PP1 binding motifs suggests ageneral mechanism wherein sequential binding to PP1c through one ofthese motifs may favor binding through the second motif and allowcatalytic function. Furthermore these motifs could also represent apredictive signature to identify new potential PP1 binding proteins. Topartially test this concept, a bioinformatic analysis was performed byusing “prose” (Katja Shuerer, IP) program in the Swissprot Release40library (October 2001) that contains 101602 non redundant proteinsequences.

As shown in Table 1A, the search for the presence of only one consensusindicates that 19.47% of the sequences in the library contain [RK]VxF or[RK]xVxF motifs and 16% contain the Bcl-2-like motif F-x-x-[RK]-x-[RK].In contrast, consistently with the notion of predictive signature,analysis of combinatorial presence of both PP1 binding motifs [RK]VxF or[RK]xVxF and F-x-x-[RK]-x-[RK] revealed only 4013 positives sequences,corresponding to 3.94% of the library (Table 1B). In addition if the F=Wequivalency (usually accepted for [RK]Vx[FW]/[RK]xVx[FW] motifs) isapplied, 4783 positives sequences corresponding to 4.7 of the librarywere found. In addition, the occasionally equivalency between R/K and Qslightly increases the number of positives proteins (5769 sequencescorresponding to 5.67%).

Furthermore, as expected, these sequences include all the known PP1interacting proteins of the Bcl-2 family (not shown). Together thisanalysis indicates that around 5% of protein sequences share the twoputative PP1 binding motifs in their sequence. Interestingly,statistical analysis suggests the distance separating the two PP1binding motifs is comprises between 0 and 180 aa for 50% of the proteins(FIG. 11). In addition, a more details analysis reveals a major peakrepresenting 22.3% of positive sequences (897 sequences for a total of4013) that correspond to a 0-50 aa interval between the two motifs.These data are consistent with the observation that a distance of 36 aaseparates the two binding motifs in BH1 and BH3 domains of Bcl-2/Bcl-wand Bcl-x_(L) proteins (not shown).

On the basis of this analysis, the Institut Pasteur is creating and willmaintain in order a new web site “PP1signature” which contains all thesequences selected from the Swissprot Release 40 library correspondingto proteins haboring the two PP1 binding motifs. By simply entering thename of a protein or an accession number or through blast analysis,everyone will immediately know if the protein has a putative PP1signature. In addition, the user will immediately identify the sequenceencompassing the two putative PP1 binding motifs.

Most characterised PP1-binding proteins share the two motifs and can beidentified in the web site (table 2). To validate this proposal of“predictive signature strategy,” four candidates were randomly selectedand their association PP1 by simple co-immunoprecipitation experimentswas confirmed (FIG. 10B).

TABLE 1A POSITIVE ONE MOTIF SEQUENCES % TOTAL LIBRARY F-x-x-R-x-R 4,0744 F-x-x[RK]-[RK] 16,260 16 [RK]-V-x-F 8,895 8.76 [RK-x-V-x-F 9,935 9.48[RK]-V-x-F or 16,273 16.02 [RK]-X-V-x-F [RK]-V-x-[FW] or 19,787 19.48[RK}-x-V-x-[FW]

TABLE 1B POSITIVE TWO MOTIFS SEQUENCES % TOTAL LIBRARY [RK]-V-x-F or1,025 1 [RK]-x-V-x-F + F-x-x-R-x-R [RK]-V-x-F or 4,013 3.94[RK]-x-V-x-F + F-x-x-R-x-R [RK]-V-x-[FW] or 4,783 4.70 [RK]-x-V-x-[FW] +F-x-x-[RK]-x-[RK] [RKQ]-V-x-F/W or 5,769 5.67 [RKQ]-x-V-x-F/W +F-x-x-[RK]-x-[RK]

TABLE 2ATwo putative PPI binding sequences in characterized PP1-interacting proteinsProtein/gene motif 1 Residues motif 2 Residues YeastG1P2 (homolog of GM) QFERKNEKLD 12-18 LIRSKSVHFDQA 216-227 6320895GIP2h/YIL045S49933 SLEFLHKPRRLS 55-60 QRSKSVUFD 191-202 *YAL014 L05146DLFNERRQRR 110-116 MPTRHNVRWEEN  45-56 REG2 6319525 RSWFKARKRRDI 152-157KPRERHIKFNDN  163-174 Mammalian Mouse PTG AAB49689 RRNFVN KLKPL 28-38NQAKKRVVFADS  56-67 TVKVKNVSFEKK 149-160 *GL CAA77083 Y LDFRNRLQTN121-130 KICVKIMVSFAND 56-67 Human R5 4885559 RHFVNKLKPLKS  48-60NQAKKRVVFADS 79-90 U5 AAC60216 TCFRPRLRGS 101-110 SQKKKRVVFADM 61-77Splicing factor PSF GEVFINKGKGF 324-334 RGRQLRVRFATH 358-369 P23246Ribosomal Protein L5 P22451 QVKFRRRREG 17-26 YFKRYQVKFRRR 12-23GRP-78 P20029 EDFKAKKKEL  615-620 RITPSYVAFTPE 61-72 Human 110pRB P06400SVFMQRLKTNILQ 758-770 IDEVKNVYFKNF 285-296 VLKVSWITFLLA 190-201AKAPs (AKAP149/AKAP220/Yotiao) LQFELRYRPV 250-255 TTKAVMFAK 141-145Hsp-90-α (Hsp 86) P07901 DLFENRKKKN  353-358 VRRVFIM 367-370Human MYPT2 4505319 FFKNEKMLY  331-340 RRGSPRVRFEDG 48-59Human I-2 CAA55475 RQFEMKRKLHY 128-138 Not detected *These two sequencescorrespond to a higher degenerated consensus (F-X-X-R/K-X-R/K/Q)Consensus F-X-X-R/K-X-R/K R/K-X-V/I-X-F Or R/K-V/I-X-F

Example 4 β Amyloid Precusor as a New PP1 Interacting Protein

Fibrillar amyloid deposits are defining pathological lesions inAlzheimer's brain diseases and are thought to mediate neuronal death.Amyloid is composed of a 39 to 42 amino acid protein fragment of theamyloid precursor protein (APP). Because depostion of fibrillar amyloidin vitro has been shown to be dependent upon the APP concentration,reducing or inhibiting the release of APP is a therapeutic target.

The β-amyloid precursor (APP) is overexpressed in PC12 cells treated byNGF, by transfection of a cDNA-encoding the β-amyloid precursor (APP)according to the methods of Sambrook et al, supra.

The cells expressing the β-amyloid precursor are then incubated with apenetrating peptide corresponding to the punitive PP1 binding site suchas the motif M1 having the sequence FXX[[RK]X[RK] and the M2 motif[RK]VX[FW] or [RKXVX[FW], where X is any amino acid.

The presence of amyloid is tested using monoclonal antibodies or a

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1-23. (canceled)
 24. A set of polypeptides having both the motifs M1 andM2 on the same polypeptide or M1 on one polypeptide and M2 on the otherpolypeptide wherein M1 and M2 are mammalian PP1 binding sequenceswherein M1 is RRNFVNKLKPL (SEQ ID NO:93) and M2 isNQAKKRWFADSTVKVKNVSFEKK (SEQ ID NO:94) or M1 is LDFRNRLQTN (SEQ IDNO:95) and M2 is KKVKKRVSFAND (SEQ ID NO:96) or M1 is RHFVNKLKPLKS (SEQID NO:97) and M2 is NQAKKRWFADS (SEQ ID NO:98) or M1 is TCFRPRLRGS (SEQID NO:99) and M2 is SQKKKRWFADM (SEQ ID NO:100) or M1 is GEVFINKGKGF(SEQ ID NO:101) and M2 is RGRQLRVRFATH (SEQ ID NO:102) or M1 isQVKFRRRREG (SEQ ID NO:103) and M2 is YFKRYQVKFRRR (SEQ ID NO:104) or M1is EDFKAKKKEL (SEQ ID NO:105) and M2 is RITPSYVAFTPE (SEQ ID NO:106) orM1 is SVFMQRLKTNILQ (SEQ ID NO:107) and M2 is IDEVKNVYFKNFVLKVSWITFLLA(SEQ ID NO:108-109) or M1 is LQFELRYRPV (SEQ ID NO:110) and M2 isTTKAVMFAK (SEQ ID NO:111) or M1 is DLFENRKKKN (SEQ ID NO:112) and M2 isVRRVFIM (SEQ ID NO:113) or M1 is FFKNEKMLY (SEQ ID NO:114) and M2 isRRGSPRVRFEDG (SEQ ID NO:115).
 25. The set of polypeptides according toclaim 24, wherein said peptides are glycosylated, acetylated,phosphorylated or amidated.
 26. A set of isolated nucleic acids encodinga set of peptides according to claim 24 or a nucleic acid sequence thathybridizes under stringent conditions to one of said nucleic acids insaid set of peptides.
 27. A vector comprising the nucleic acidsaccording to claim
 26. 28. A method of identifying a PP1-interactingpolypeptide or protein, comprising: detecting in the sequence saidpolypeptide or protein, the presence of two PP1-binding motifs M1 and M2on the same polypeptide, wherein the motif M1 and M2 is chosen from oneof the motif pairs in claim 24; and confirming said identified PP-1interacting polypeptide or protein using a biochemical test.
 29. Themethod of claim 28, wherein said biochemical test consists incoimmunoprecipitating said identified PP-1 interacting polypeptide orprotein and PP1.
 30. A method of screening compounds that inhibit orenhance PP1 activity or change its localization by interaction or thatinteract with PP1 regulators, comprising; (a) immobilizing peptidesaccording to claim 24 on a support; and (b) testing the interaction ofsaid compounds with the peptides of step (a).
 31. A method of screeningcompounds that interact with PP1, comprising: i. obtaining antibodies topeptides according to 24 1 and ii. testing the interaction of saidcompound with said antibodies.
 32. A pharmaceutical compound comprisingthe set of peptides according to claim
 24. 33. A pharmaceuticalcomposition comprising a nucleic acid encoding the set of peptidesaccording to claim
 24. 34. A pharmaceutical composition comprising thevector of claim
 27. 35. A method for inhibiting in vitro the interactionbetween PP1 c and Bc1XL, comprising a step of adding a set of peptideshaving both the motifs M1 and M2 on the same peptide, wherein the set ofpeptides comprises one of the M1 and M2 motif pairs in claim
 24. 36. Akit comprising antibodies to the set of peptides according to claim 24and reagents for interpreting the interaction.
 37. A method of treatingor preventing diabetes, high blood pressure, a neurological disorder, aviral disease or a microbial infection associated with PP1 said methodcomprising administering to a patient in need of such treatment the setof polypeptides according to claim
 24. 38. The method according to claim37, wherein the neurological disorder is Parkinson's disease orAlzheimers disease.
 39. The method according to claim 37, wherein theviral disease is HIV-1.
 40. Antibodies to the peptides according toclaim
 24. 41. Antibodies according to claim 40, wherein said antibodiesare monoclonal antibodies.
 42. The set of polypeptides according toclaim 24, wherein there are two or more M1 and M2 motifs on the samepolypeptide.
 43. The set of polypeptides according to claim 42, whereinthere are 2 to 5, M1 and M2 motifs on the same polypeptide.
 44. The setof polypeptides according to claim 42, wherein there are 2 to 10, M1 andM2 motifs on the same polypeptide.
 45. The set of polypeptides accordingto any one of claim 42, wherein said M1 and M2 motifs have a spacerbetween them.
 46. The set of polypeptides according to claim 45, whereinthe spacer is selected from a sequence of amino acids, a hydrocarbonchain or a chemical entity which connects the at least two motifs. 47.The set of polypeptide according to claim 46, wherein said amino acidspacer has between 1 to 50 amino acids.
 48. The set of polypeptideaccording to claim 46, wherein amino acid spacer has 36 amino acids. 49.The set of polypeptide according to claim 46, wherein said spacercontains regulatory sequences between the motifs.